The structure, function and expression analysis of the nodulin 26-like intrinsic protein subfamily of plant aquaporins in tomato

The nodulin 26-like intrinsic protein (NIP) family belonging to a group of aquaporin proteins is unique to plants. NIPs have a wide of transport activities and are involved in developmental processes and stress tolerance. The well reported Lsi1 and Lsi6 belonging to NIP III were characterized as Si transporters. However, except Lsi1 and Lsi6, most NIPs remain unknown. Here, we identified 43 putative aquaporins in tomato. We found there are 12 NIPs, including 8 NIP I proteins, 3 NIP II proteins, and 1 NIP III protein among the 43 aquaporins. Also, there are two Si efflux transporters SlLsi2-1 and SlLsi2-2 identified by using Lsi2 proteins from other species. By analysing the phylogenetic relationships, conserved residues and expression patterns, we propose that three NIP I members (SlNIP-2, SlNIP-3 and SlNIP-11) may transport water, ammonia, urea, and boric acid, and contribute to pollen development. Three NIP II proteins (SlNIP-7, SlNIP-9 and SlNIP-12) may be boric acid facilitators, and affect plant growth and anther development. Overall, the study provides valuable candidates of Si transporters and other NIP proteins to further explore their roles in uptake and transport for silicon, boron, and other substrates in tomato.

The chromosomal localization of 45 Lsi1/Lsi2/Lsi6 homologous genes in tomato. The chromosomal localizations of 45 tomato Lsi1/Lsi2/Lsi6 homologues were determined to visualize their genomic position information. They were distributed on 10 of 12 chromosomes except for chromosome 4 and chromosome 7 (Fig. 2). Among them, eight genes were mapped to chromosome 3, 6 and 10; two on chromosome 5 and 9, three on chromosome 12, four on chromosome 1 and 8, five on chromosome 2; there was only one gene on chromosome 11 (Fig. 2). It should be noted that gene duplication may occur among these tomato Si transporter homologues, since the loci Solyc10g054790, Solyc10g054800, Solyc10g054810 to Solyc10g054820 are found next to each other on chromosome 10. Also the gene pairs Solyc02g071910/Solyc02g071920, Solyc09g007760/ Solyc09g007770 were found on chromosome 2 and chromosome 9, respectively. Interestingly, Solyc10g054790,  www.nature.com/scientificreports/ Solyc10g054800, Solyc10g054810 and Solyc10g054820 were clustered together in Clade I, Solyc02g071910/ Solyc02g071920 were grouped together in Clade III, and Solyc09g007760/Solyc09g007770 were clustered together in Clade V (Fig. 1), which showed high similarity based on protein sequences, indicating they may share common functions with each other.
Analysis of exon-intron structure. The exon and intron information of 45 tomato Lsi1/Lsi2/Lsi6 homologues were searched in SGN database and sorted by GSDS. The color bar on the left side of the gene name represents the clades of these genes in the evolutionary tree. Exon-intron analysis showed that the size and the number of the exons are highly conserved within each clade, but significantly different among the clades. Most members of the 45 tomato Lsi1/Lsi2/Lsi6 homologous genes contained three to five exons, but a few contained less than three or more than five exons. Members of Clade I and Clade II are characterized by three exons. Most members of Clade III have five exons. The majority of the members of Clade IV features three exons, while most members of Clade V contain four exons ( Supplementary Fig. S1A,B).
Gene structure and motif composition analysis. Motifs are highly conserved amino acid residues in homologous proteins, which may play important roles in the structure and function of active proteins. The conserved motifs of these 45 tomato Lsi1/Lsi2/Lsi6 homologues were analyzed by MEME. Ten conserved motifs were identified ( Supplementary Fig. S1C). Among them, Motif 9 only exists in the members of Clade II and this clade does not contain any other motifs, which might contribute to the functional divergence of Lsi2 proteins in Clade II. It is reasonable Lsi2 was ion transporter protein, differing from NIP proteins. Motifs 1, 2 and 3 were found in Clade III that clustered by Lsi1/Lsi6 proteins, which were also shared in Clade I, IV and V. Interestingly, members of Clade I and V, and some members of Clade IV, but not any members of Clade III, also shared Motif 7. Motif 8 exists in all members of Clade V and some members of Clade I; in addition, members in Clade I solely contain Motifs 5, 6 and 10, and members in Clade V uniquely possess Motif 4 ( Supplementary Fig. S1A,C). These observations suggest genes in Clade I, IV and V may have evolved from genes in Clade III, sharing common motifs (Motifs 1, 2 and 3) and similar functions with members in Clade III, and further gained some additional motifs (such as Motif 7) and corresponding functional diversification afterwards.

Prediction of physicochemical properties and identification of conserved domain. It is well
known that NIPs of aquaporin subfamily were characterized for six transmembrane domains (TMDs) connected by five loops (loop A-loop E) 38 , two highly conserved NPA (asparagine-proline-alanine) motifs on loop B and loop E, an ar/R selectivity filter and Froger's positions 39 . Sequence alignments of twelve tomato NIPs were performed with ClustalX2 (Fig. 4). The TMDs of each protein were predicted by TMHMM2.0 online tool, the positions of TMDs are marked with gray areas, and the numbers of TMDs of each protein are recorded in Table 1.
All the identified Si transporters of Lsi1/Lsi6 in other species contain six TMDs, and most of the twelve tomato NIPs also contain six TMDs. However, there is an exception for SlNIP-10 and SlNIP-11, which possess four and three TMDs, respectively. The second TMD in SlNIP-10 is incomplete, while SlNIP-11 missed the fourth, fifth and sixth TMDs (Table 1). For the members of aquaporin family, NPA domains will generate electrostatic repulsion to protons, and then form a channel, while ar/R selectivity filter and Froger's positions will affect the specific substrate of the channel. All Si transporters identified in other species have two NPA domains, only with an exception for CsLsi1, which have NPV on Loop E with the alanine (A) in the third position replaced by threonine (T). However, NPA motif changed into NPV does not disturb Si transport activity of CsLsi1 35 . As for tomato, 11 of the 12 tomato NIPs all carried two NPA/S/V/T motifs, except for SlNIP-11 only having one NPA motif on Loop B. A specific length of 108 AA between the two NPA domains is a necessary and selectivity feature for Si among all Si-transporting plants 29 . These already validated Si-transporting Lsi1 proteins all presented 108 AA residues, such as CsLsi1, HvLsi1, TaLsi1, SbLsi1, CmLsi1, ZmLsi1 and OsLsi1. Whereas most of the tomato NIPs have 109 residues in the spacing, and only SlNIP-7 and SlNIP-9 have 108 AA. Moreover, SlNIP-8 has 112 AA and SlNIP-10 has 115 AA between the two NPA domains. However, the spacing itself is unlikely to be required for Si permeability. Supporting evidences showed that SlLsi1/SlNIP-1 possesses 109 AA in the spacing and shows transport activity for Si 30 .
For most members of NIP III subgroup from various species, including SlNIP-1 from tomato, four amino acids from helix 2 (H2), helix 5 (H5), and loop E (LE1 and LE2) that constitute ar/R selectivity filter are GSGR, except for CsLsi6, where a small G in the H2 is substituted with the bulkier C residue (Table 1). However, the residue at the H2 position is not critical for Si transport. For example, when glycine (G) was substituted by alanine www.nature.com/scientificreports/ (A) at H2 of OsLsi1, the transport activities for Si were unaffected 40 . Compared to residue at H2, residue at H5 is required for Si transport activity. When serine (S) at the H5 position was substituted by isoleucine (I), and the transport activity of OsLsi1 for Si was totally lost 40 . In addition, when both residues at the H2 and H5 were substituted, Si transport activities of OsLsi1 as well as transport activities for other solutes are completely lost. Most of tomato NIPs belong to NIP I subgroup possessing an ar/R filter consisting of W, V, A and R, except SlNIP-6 with ar/R selectivity filters of WIAR, and SlNIP-10 and SlNIP-11 whose ar/R selectivity filters are incomplete. For SlNIP-7, SlNIP-9 and SlNIP-12, which belong to the NIP II subgroup, their ar/R selectivity filters are SIAR, TIAR, and AVGR, respectively. It proposed us to speculate that SlNIP-6, SlNIP-7 and SlNIP-9 with I at H5, and SlNIP-10 and SlNIP-11with incomplete ar/R selectivity filter might have no transport activities for Si. It is worth testing the transport activities of SlNIP-1, SlNIP-2, SlNIP-3, SlNIP-4, SlNIP-5, SlNIP-8 with ar/R selectivity filter consisting of WVAR and SlNIP-12 with ar/R selectivity filter consisting of AVGR.  www.nature.com/scientificreports/ (S or T), which are usually the sites of phosphorylation of proteins. This residue may be responsible for the post-transcriptional modification and regulation of protein activity. However, the amino acid composition of Position 5 differs from SlNIP-1 to these already identified Si transporters. Position 5 is a nonaromatic residue in SlNIP-1, and this residue is aromatic in these already identified Si transporters (Table 1). Furthermore, within tomato NIPs, residues at Position 1 are also different between SlNIP-1 and members of tomato NIP I and NIP II subgroups. Residue at Postion 1 is an aromatic residue in NIP I and NIP II members, and this residue is not aromatic in SlNIP-1. Whether the variant residues at Position 1 and Position 5 in the Froger's positions affect transport activities of NIPs or not, which need to be experimentally proved in further studies. The predicted results of physical and chemical properties of these Si transporters as well as members of tomato NIP I and NIP II are recorded in Table 1. Except SlNIP-11 (137 AA and 14.86, respectively), the length of the encoded proteins mostly ranged from 231 to 345 amino acids, and their predicted molecular mass ranged from ~ 25.45 to 37.22 kDa. The isoelectric points of all 24 Si transporters ranged from 6.05 to 9.40, and the total average hydrophobicity of protein is between 0.33 and 0.75. The subcellular localization of these Si transporters and NIPs is predicted by WoLF online tool and recorded in Table 1. It is found that most of these proteins are located in plasma membrane, some are located in chloroplast membrane, vacuole membrane or endoplasmic reticulum. The difference between localizations may be related to the functional differences, and individual proteins may be involved in the transport of substances between plasma membrane and organelles.
Tissue-specific expression analysis. To explore the functions of tomato NIP genes as well as the two SlLsi2 genes, we firstly investigated their expression atlas based on the RNA-seq data derived from the TOMATO FUNCTIOPNAL GENOMICS DATABASE in 11 various tissues of tomato including whole root, hypocotyl, cotyledons, young leaves, mature leaves, vegetative meristems, young flower buds, flowers at anthesis (0 DPA), 10 days post anthesis fruit (10 DPA), 20 days post anthesis fruit (20 DPA) and ripening fruit (33 DPA) (Fig. 5). SlNIP-7 and SlNIP-2 exhibited high and organ-specific expression pattern. SlNIP-7 was highly expressed in all examined tissues, especially in root, indicating it may function in root. SlNIP-2 was preferentially expressed highly in anthesis flowers. SlNIP-5, SlNIP-9 and SlLsi2-1 were constitutively expressed at a high level in nearly all tested tissues, and four genes (SlNIP-8, SlNIP-10, SlNIP-11 and SlNIP-12) were expressed with very low transcript abundance. SlNIP-3 was preferentially expressed in anthesis flowers, while SlNIP-6 and SlLsi2-2 was spe- www.nature.com/scientificreports/ cially expressed in root. A similar expression pattern was observed on SlNIP-4 and SlNIP-5 genes, which showed relatively higher expression levels in root, hypocotyl, cotyledons, and fruit (20 DPA), but a lower level of expression in ripening fruit. SlNIP-1 showed relatively higher expression levels in root, hypocotyl, cotyledons, anthesis flowers, immature fruit, but relatively lower expression levels in young leaves, mature leaves, vegetative meristems, young flower buds and ripening fruit, indicating SlNIP-1 may function during fruit development (Fig. 5).
To further explore the possible functions of tomato NIP genes, organ-specific expression profiles of these genes were further verified by RT-qPCR. We used 6 different tissues (root, stem, leaf, flower, mature green fruit, and red mature fruit). As shown in Fig. 6, expression patterns of most of these genes were consistent with the RNA-seq data in Fig. 5. Consistent with previous report, the already identified Si influx transporter SlNIP-1 was mainly expressed in root relative to leaf, and Si efflux transporter SlLsi2-2 was specific to root (Fig. 6 30 ). Inconsistent with previous report 30 , another efflux transporter SlLsi2-1 was expressed mainly in leaf but not in root, it may be due to SlLsi2-1 was not a consecutive expression gene but an induced gene. Six genes (SlNIP-2, SlNIP-3, SlNIP-8, SlNIP-9, SlNIP-11 and SlNIP-12) had similar expression patterns and demonstrated a high expression in flower. SlNIP-4 was expressed strongly in root and stem, while SlNIP-10 was expressed intensely in stem and flower. SlNIP-5 was expressed significantly in root and green mature fruit, weakly in stem and red mature fruit, and moderately in leaf and flower, whereas, SlLsi2-1 was expressed highly in stem and leaf, weakly in root and red mature fruit, and moderately in flower and green mature fruit. SlNIP-6 like SlLsi2-2 displayed root-specific expression patterns. SlNIP-7 was constitutively expressed at a high level in almost all tested tissues, and showed relatively higher expression levels in root. Above all, different members of the tomato NIP genes showed distinct expression patterns, indicating these genes may function in many aspects of tomato growth and development.
Promoter analysis. Specific cis-acting elements can combine with particular transcription factors to regulate downstream gene transcription and expression. In order to determine the promoter cis-acting elements of 14 tomato Si transporter homologous proteins, the 2000 bp sequences upstream of the start site (ATG) of these genes were downloaded as putative promoters from SGN database. The promoter sequences were analyzed by using PlantCARE online tool, and the identified cis-acting elements were sorted into three groups related to plant growth and development, hormone response and biotic/abiotic stresses (Fig. 7). Cis-acting elements related to plant growth and development included endosperm expression element (GCN4_motif), circadian rhythm regulation element (circadian), zein metabolism regulation element (O 2 -site), meristem expression element (CAT-box), seed-specific regulation element (RY-element) and mesophyll cell differentiation element (HD-Zip1). There are only 0-3 cis-acting elements of this group in promoters of all 14 tomato Si transporter homologous genes, which indicates that these genes may be less induced by signaling from the specific growth and development stage of plants. Cis-acting elements belonging to hormone response included salicylic acid responsive element (TCA-element), abscisic acid (ABA) responsive element, methyl jasmonate responsive element (CGTCA-motif, TGACG-motif), ethylene responsive element (ERE), auxin responsive element (TGAelement, AuxRR-core) and gibberellin responsive element (TATC-box, GARE-motif, P-Box), which has 3-14 cis-acting elements of this group in the promoters of 14 tomato Si transporter homologues, indicating that these genes are possibly sensitive to plant hormones. Eleven tomato Si transporter homologues except SlNIP-2, SlNIP-4 and SlNIP-7 all have ABA responsive elements, while eight tomato Si transporter homologues except SlNIP-3, SlNIP-6, SlNIP-9, SlNIP-11, SlNIP-12 and SlLsi2-1 all have ethylene responsive element (ERE). ABA and

Subcellular localization of SlNIP-1 and its mutated proteins. Plasma membrane location is required
for the uptake of Si by Lsi1 transporters. Functional Lsi1 Si influx transporters are unexceptionally localized in the plasma membrane, such as OsLsi1 13 , HvLsi1 33 , ZmLsi1 34 , TaLsi1 22 , SlLsi1 29 , CsLsi1 35 , and NsLsi1 41 . Subcellular localization can largely affect the Si transport activities. CmLsi1 from the bloomless rootstock that was localized at the plasma membrane have transport activity, whereas the one from the bloomless rootstock was localized at the endoplasmic reticulum and had no transport activity for Si. In order to verify the subcellular localization of tomato SlNIP-1, we cloned the 849 bp CDS sequence of SlNIP-1, and introduced it into the vector pGWB5 to obtain the GFP fusion vector SlNIP-1-pGWB5 (35S::SlNIP-1-GFP). The vector was transformed into Agrobacterium tumefaciens GV3101 to generate transgenic Arabidopsis plants by floral dip method. T 1 transgenic Arabidopsis plants were used for observing GFP fluorescence signal. Consistent with WoLF's prediction in Table 1 and previous report 29 , SlNIP-1 protein was localized in plasma membrane (Fig. 8). A 108-AA spacing between NPA domains is essential to Si influx transport activity for SlLsi1 29 . Predicted by PROVEAN, we found five site deletions (140 V, 141 T, 142 K, 143 N and 144 V) result in neutral effects. Then, the localization of variant SlNIP-1 mutants of single amino acid deletion was confirmed by transgenic plants. As the results showed that, like SlNIP-1, these five mutated SlNIP-1 proteins (SlNIP-1Δ140V, SlNIP-1Δ141T, SlNIP-1Δ142K, SlNIP-1Δ143N, and SlNIP-1Δ144V) with a spacing of 108 AA were also located on the plasma membrane (Fig. 8).
In 2015, Deshmukh et al. had reported that deletion 140 V in SlNIP-1 exhibited Si permeability relative to the native SlNIP-1 protein 29 . Thus, the function of the other four SlNIP-1 mutants need to be tested.

Discussion
Tomato Si transporter homologues SlNIP-1 to SlNIP-12 belong to NIP subfamily (Figs. 1, 3). Six of the twelve proteins with ar/R filter consisting of Trp (H2), Val (H5), Ala (LE1), and Arg (LE2), WVAR, as well as SlNIP-6 with WIAR, belong to the NIP I subgroup, whereas SlNIP-7 with SIAR, SlNIP-9 with TIAR, and SlNIP-12 with AVGR were classified into NIP II group 42,43 . Additionally, SlNIP-1 with GSGR belong to NIP III group 28 . Compared to NIP III subgroups, which have small amino acid residues Gly in H2 and Ser in H5; members of NIP I subgroups have two bulky residues Trp and Val in H2 and H5 positions, respectively 23 . So, considering the composition of ar/R filter, we propose that most members of tomato NIPs possessing WVAR (WIAR in SlNIP-6) do not have transport activities for silicic acid, but may transport small molecules of water, glycerol www.nature.com/scientificreports/ and lactic acid as members in NIP I. To support the propose, one study showed that replacement of AIGR by WIGR endows AtNIP6;1 with water transport activity, suggesting the possible water-transport activity as well as glycerol transport of SlNIP-6 which was presented by WIAR of the ar/R region. The Arabidopsis NIP II subgroup has three member genes: AtNIP5;1, AtNIP6;1, and AtNIP7;1 23 , and each of them has an ortholog in tomato, represented by SlNIP-7, SlNIP-9 and SlNIP-12, respectively 43 . Current study further confirmed the remarkable orthologous relationships between AtNIP5;1, AtNIP6;1, AtNIP7;1 and SlNIP-7, SlNIP-9, SlNIP-12. Firstly, structural similarity is within pore determinant regions, such as SlNIP-7 and AtNIP5;1 share the a/R filter of SIAR; SlNIP-9 and AtNIP6;1 share the a/R filter of TIAR; SlNIP-12 together with AtNIP7;1 share the a/R filter of AVGR (Table 1). Secondly, similar tissue expression profiles, RT-qPCR combined with RNA-seq data showed that SlNIP-7 was mainly expressed in the roots, SlNIP-9 was constitutively expressed in all examined tissues with high expression level in stem, and SlNIP-12 was specifically expressed in flower (Fig. 5), which were similar to previous studies [43][44][45] . Thirdly, similar to previous observations with AtNIP5;1 45 , AtNIP6;1 44 and AtNIP7;1 43 , SlNIP-7 and SlNIP-9 were predicted to be localized on plasma membrane, and SlNIP-9 was predicted to be localized on vacuole membrane ( Table 1). As AtNIP5;1, AtNIP6;1, and AtNIP7;1 were reported to mediate boron uptake and contribute to the development of root, rosette leaves, inflorescences and pollen grain 27,43,44 . In summary, the results suggested SlNIP-7, SlNIP-9 and SlNIP-12 might act as major channel proteins mediating boric acid transport. It will be important to investigate the substrate specificity of the SlNIP-7, SlNIP-9 and SlNIP-12 and the possible involvement of SlNIP-7, SlNIP-9 and SlNIP-12 in plant growth and development.
In addition to ar/R selectivity filter, NPA motif and inter-NPA distance can also affect the Si permeability of Lsi1 channels. It has been reported that two NPA motifs localized at loop B and loop E form part surface of the narrow aqueous pore 46 . Residues composed of NPA motifs influence the size of constriction filter, and then the specificity of substrates. For example, the pore diameter of the NPA regions of AtNIP6;1 is narrower than that of Nodulin 26, which may result from a larger Val substitution for the small Ala in the second NPA motif (loop E) of AtNIP6;1 compared with Nodulin 26 26 . Most members of the tomato NIPs have two conserved NPA motifs, while the first NPA motif in SlNIP-4 and SlNIP-7 was changed to NPS. In addition, the second NPA motif in SlNIP-7 was also replaced by NPV. Moreover, the second motif in SlNIP-9 and SlNIP-10 was changed to NPV and NPT, respectively. However, it seems that the second NPA motif is not a crucial factor for the substrate specificity of NIP proteins. In the case of AtNIP6;1, a substitution of Ala for Val in the second NPA motif has little effect on the transport selectivity of AtNIP6;1 26 .
An inter-NPA distance of 108-AA is a common feature that is critical for the function of Si transporters. SlNIP-1/SlLsi1 owned an ar/R filter composing of GSGR belonging to the NIP III group and might be a Si transporter, but 109 AA in the inter-NPA region of SlNIP-1 may cause the loss function of Si transport activities. However, Si permeability of SlLsi1 is still controversial 29,30 . In addition to the precise space between the NPA domains, other molecular determinants can affect the function of Lsi1 transporters. NsLsi1 from tobacco possessing typical molecular signatures, a GSGR selectivity filter and a 108 AA inter-NPA space, were found to be Si-impermeable 41 . Further investigation showed that Si transport activities of NsLsi1 can be compromised by P125F substitution. P125F substitution increased plasma membrane localization of NsLsi1 P125F compared to NsLsi1 WT , thus enhanced the transport capacity 41 . It was also reported that analysis of boric acid permeability of AtNIP7;1 by expressing AtNIP7;1 in Xenopus laevis oocytes and proteoliposomes yielded contrasting results, explaining by some factors influencing the pore structures of the AtNIP7;1 changed in this two systems 43 . As for the case of SlNIP-1, other than the molecular characteristics of protein sequences, determining factors, such as biochemical properties as well as posttranslational modification, which affect the function of SlNIP-1 may be changed due to different analysis systems and methods. Besides, deletion 140 V in SlLsi1/SlNIP-1 restored its Si permeability 29 . In the future, the activity of SlNIP-1Δ141T, SlNIP-1Δ142K, SlNIP-1Δ143N, and SlNIP-1Δ144V are worth testing. Finally, definition of the in vivo transport properties of SlNIP-1 will require further detailed investigation.
Tissue-specific expression pattern reflects the function of genes to some extent. We conducted RNA-seq and RT-qPCR analysis, discovering some interesting trends and expression patterns of 14 tomato Si transporter homologues (Figs. 5, 6). SlNIP-1 and SlLsi2-2 were mainly expressed in root relative to leaf, which is consitent with their function reported in previous study 30 . SlLsi2-1 was found mainly expressed in leaf but not root, which did not match previous results 30 . This discrepancy may reflect that SlLsi2-1 was not expressed constantly but can be induced by some stimulation. The high expression pattern in root was also found in SlNIP-4, SlNIP-5, and SlNIP-6, implying their roles in root. Indeed, AtNIP2;1 from Arabidopsis was a root-specific expression gene, and it was proved to be a lactic acid efflux channel and is necessary for an optimum response to low oxygen stress 47 . Thus, considering SlNIP-4, SlNIP-5 and SlNIP-6 both belong to the NIP I family and with similar root-specific expression as AtNIP2;1, we propose that SlNIP-4, SlNIP-5 and SlNIP-6 may be involved in abiotic stress response in root.
SlNIP-2, SlNIP-8, and SlNIP-11 exhibited an almost undetectable low expression level in most examined tissues but show high expression levels in flower, suggesting these genes may play specific roles in flower. Similarly, SlNIP-3 and SlNIP-10 were also mainly expressed in flower. Polygenetic tree indicated that SlNIP-2, SlNIP-3 and SlNIP-11 were highly homologous with AtNIP4;1 and AtNIP4;2. It has been reported that AtNIP4;1 and AtNIP4;2 were expressed in flower specific to pollen grains and pollen tubes, and characterized as important regulators for the proper pollen development, pollen germination, and pollen tube growth 48 . Similar to AtNIP4;1 and AtNIP4;2, SlNIP-2, SlNIP-3 and SlNIP-11 may participate in the male gametophyte development.

Conclusions
There are 43 aquaporins including 8 NIP I proteins, 3 NIP II proteins, and 1 NIP III protein in tomato. SlNIP-1 was the reported Si influx transporters SlLsi1. Mutated SlNIP-1 proteins of single amino acid deletion-SlNIP-1Δ141T, SlNIP-1Δ142K, SlNIP-1Δ143N, and SlNIP-1Δ144V-resembled the native SlNIP-1 showing plasma membrane localization. Though polygenetic tree analysis, conserved structure analysis, and gene expression patterns analysis, we predict SlNIP-7, SlNIP-9, SlNIP-12 may be boric acid facilitators, and SlNIP-2, SlNIP-3 and SlNIP-11 may be involved in the pollen development. Considering the important biological functions of NIP subfamily of plant aquaporins, further analysis of the functions of these genes in specific tissue is deserved.
In addition, we also propose that functional identification of SlNIP-1, SlLsi2-1 and SlLsi2-2 should be obtained through transgenic plants and furthermore experimental studies. Phylogenetic tree construction and chromosome location. www.nature.com/scientificreports/ The plant materials were well used for research project and comply with relevant institutional, national, and international guidelines and legislation.

RNA extraction and quantitative RT-PCR (qRT-PCR) analysis. TRIzol reagent (Invitrogen) was
used for RNA extraction. For quantitative real-time polymerase chain reaction (qRT-PCR) analysis, one microgram of DNA-free RNA was transcribed into first strand cDNA by Prime-Script RT Master Mix (TaKaRa). The qRT-PCR was carried out with the UVP ChemStudio (analyticjena) using TB Green Premix Ex Taq (TaKaRa). The reaction conditions were 95 °C for 30 s, and 40 cycles at 95 °C for 5 s, 60 °C for 30 s. Expression levels of target genes were normalized relative to ACTIN2 gene. Primers used to quantify gene expression levels are listed in Supplementary Table S4. Each reaction was performed with three biological replicates.
Cis-elements analysis. Regions 2000 bp upstream of the start codon (ATG) of each gene were downloaded in the tomato genome database SGN as the predicted promoter sequences. Cis-acting elements of the promoters were predicted by PlantCARE (http:// bioin forma tics. psb. ugent. be/ webto ols/ plant care/ html/).
Microscopy observations. For subcellular localization analysis, roots of 10-day-old T 1 transgenic plants with corresponding constructs were observed and imaged under a laser-scanning confocal microscope (Olympus fluoview FV3000). For imaging GFP, the 488-nm lines of the laser were used for excitation, and emission was detected at 510 nm.

Data availability
The amino acid sequences analyzed in the current study are available in the Sol Genomics Network repository (https:// solge nomics. net/), the NCBI database (https:// www. ncbi. nlm. nih. gov/), the Arabidopsis Information Resource (TAIR) (http:// www. arabi dopsis. org/) and the rice genome annotation database (http:// rice. uga. edu/), respectively. The accession numbers are included in the Supplementary Tables. License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.